Cells and non-human organisms containing predetermined genomic modifications and positive-negative selection methods and vectors for making same

ABSTRACT

Positive-negative selector (PNS) vectors are provided for modifying a target DNA sequence contained in the genome of a target cell capable of homologous recombination. The vector comprises a first DNA sequence which contains at least one sequence portion which is substantially homologous to a portion of a first region of a target DNA sequence. The vector also includes a second DNA sequence containing at least one sequence portion which is substantially homologous to another portion of a second region of a target DNA sequence. A third DNA sequence is positioned between the first and second DNA sequences and encodes a positive selection marker which when expressed is functional in the target cell in which the vector is used. A fourth DNA sequence encoding a negative selection marker, also functional in the target cell, is positioned 5′ to the first or 3′ to the second DNA sequence and is substantially incapable of homologous recombination with the target DNA sequence. The invention also includes transformed cells containing at least one predetermined modification of a target DNA sequence contained in the genome of the cell. In addition, the invention includes organisms such as non-human transgenic animals and plants which contain cells having predetermined modifications of a target DNA sequence in the genome of the organism.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a Continuation application Ser. No. 08/461,827, filed on Jun. 5,1995, now U.S. Pat. No. 5,627,059, the disclosure of which isincorporated by reference.

This application is a 37 CFR §1.53 divisional application ofcommonly-owned, application Ser. No. 08/014,083, filed Feb. 4, 1993, nowU.S. Pat. No. 5,464,764, which is a continuation of Ser. No. 08/084,741,Jun. 28, 1993, now U.S. Pat. No. 5,487,992, which is a continuation ofSer. No. 07/397,707, filed Aug. 22, 1989, now abandoned. These documentsare hereby incorporated by reference for all purposes.

TECHNICAL FIELD OF THE INVENTION

The invention relates to cells and non-human organisms containingpredetermined genomic modifications of the genetic material contained insuch cells and organisms. The invention also relates to methods andvectors for making such modifications.

BACKGROUND OF THE INVENTION

Many unicellular and multicellular organisms have been made containinggenetic material which is not otherwise normally found in the cell ororganism. For example, bacteria, such as E. coli, have been transformedwith plasmids which encode heterologous polypeptides, i.e., polypeptidesnot normally associated with that bacterium. Such transformed cells areroutinely used to express the heterologous gene to obtain theheterologous polypeptide. Yeasts, filamentous fungi and animal cellshave also been transformed with genes encoding heterologouspolypeptides. In the case of bacteria, heterologous genes are readilymaintained by way of an extra chromosomal element such as a plasmid.More complex cells and organisms such as filamentous fungi, yeast andmammalian cells typically maintain the heterologous DNA by way ofintegration of the foreign DNA into the genome of the cell or organism.In the case of mammalian cells and most multicellular organisms suchintegration is most frequently random within the genome.

Transgenic animals containing heterologous genes have also been made.For example, U.S. Pat. No. 4,736,866 discloses transgenic non-humanmammals containing activated oncogenes. Other reports for producingtransgenic animals include PTC Publication No. W082/04443 (rabbitβ-globin gene DNA fragment injected into the pronucleus of a mousezygote); EPO Publication No. 0 264 166 (Hepatitis B surface antigen andtPA genes under control of the whey acid protein promotor for mammarytissue specific expression); EPO Publication No. 0 247 494 (transgenicmice containing heterologous genes encoding various forms of insulin);PTC Publication No. W088/00239 (tissue specific expression of atransgene encoding factor IX under control of a whey protein promoter);PTC Publication No. W088/01648 (transgenic mammal having mammarysecretory cells incorporating a recombinant expression system comprisinga mammary lactogen-inducible regulatory region and a structural regionencoding a heterologous protein); and EPO Publication No. 0 279 582(tissue specific expression of chloramphenicol acetyltrans-ferase undercontrol of rat β-casein promoter in transgenic mice). The methods andDNA constructs (“transgenes”) used in making these transgenic animalsalso result in the random integration of all or part of the transgeneinto the genome of the organism. Typically, such integration occurs inan early embryonic stage of development which results in a mosaictransgenic animal. Subsequent generations can be obtained, however,wherein the randomly inserted transgene is contained in all of thesomatic cells of the transgenic animals.

Transgenic plants have also been produced. For example, U.S. Pat. No.4,801,540 to Hiatt, et al., discloses the transformation of plant cellswith a plant expression vector containing tomato polygalacturonase (PG)oriented in the opposite orientation for expression. The anti-sense RNAexpressed from this gene is capable of hybridizing with endogenous PGmRNA to suppress translation. This inhibits production of PG and as aconsequence the hydrolysis of pectin by PG in the tomato.

While the integration of heterologous DNA into cells and organisms ispotentially useful to produce transformed cells and organisms which arecapable of expressing desired genes and/or polypeptides, many problemsare associated with such systems. A major problem resides in the randompattern of integration of the heterologous gene into the genome of cellsderived from multicellular organisms such as mammalian cells. This oftenresults in a wide variation in the level of expression of suchheterologous genes among different transformed cells. Further, randomintegration of heterologous DNA into the genome may disrupt endogenousgenes which are necessary for the maturation, differentiation and/orviability of the cells or organism. In the case of transgenic animals,gross abnormalities are often caused by random integration of thetransgene and gross rearrangements of the transgene and/or endogenousDNA often occur at the insertion site. For example, a common problemassociated with transgenes designed for tissue-specific expressioninvolves the “leakage” of expression of the transgenes. Thus, transgenesdesigned for the expression and secretion of a heterologous polypeptidein mammary secretory cells may also be expressed in brain tissue therebyproducing adverse effects in the transgenic animal. While the reasonsfor transgene “leakage” and gross rearrangements of heterologous andendogenous DNA are not known with certainty, random integration is apotential cause of expression leakage.

One approach to overcome problems associated with random integrationinvolves the use gene of targeting. This method involves the selectionfor homologous recombination events between DNA sequences residing inthe genome of a cell or organism and newly introduced DNA sequences.This provides means for systematically altering the genome of the cellor organism.

For example, Hinnen, J. B., et al. (1978) Proc. Natl. Acad. Sci. U.S.A.,75, 1929-1933 report homologous recombination between a leu2+ plasmidand a leu2⁻ gene in the yeast genome. Successful homologoustransformants were positively selected by growth on media deficient inleucine.

For mammalian systems, several laboratories have reported the insertionof exogenous DNA sequences into specific sites within the mammaliangenome by way of homologous recombination. For example, Smithies, O., etal. (1985) Nature, 317, 230-234 report the insertion of a linearizedplasmid into the genome of cultured mammalian cells near the β-globingene by homologous recombination. The modified locus so obtainedcontained inserted vector sequences containing a neomycin resistancegene and a sup F gene encoding an amber suppressor t-RNA positionedbetween the δ and β-globin structural genes. The homologous insertion ofthis vector also resulted in the duplication of some of the DNA sequencebetween the δ and β-globin genes and part of the β-globin gene itself.Successful transformants were selected using a neomycin relatedantibiotic. Since most transformation events randomly inserted thisplasmid, insertion of this plasmid by homologous recombination did notconfer a selectable, cellular phenotype for homologous recombinationmediated transformation. A laborious screening test for identifyingpredicted targeting events using plasmid rescue of the supF marker in aphage library prepared from pools of transfected colonies was used. Sibselection utilizing this assay identified the transformed cells in whichhomologous recombination had occurred.

A significant problem encountered in detecting and isolating cells, suchas mammalian and plant cells, wherein homologous recombination eventshave occurred lies in the greater propensity for such cells to mediatenon-homologous recombination. See Roth, D. B., et al. (1985) Proc. Natl.Acad. Sci. U.S.A., 82 3355-3359; Roth, D. B., et al. (1985), Mol. Cell.Biol., 5 2599-2607; and Paszkowski, J., et al. (1988), EMBO J., 7,4021-4026. In order to identify homologous recombination events amongthe vast pool of random insertions generated by non-homologousrecombination, early gene targeting experiments in mammalian cells weredesigned using cell lines carrying a mutated form of either a neomycinresistance (neo^(r)) gene or a herpes simplex virus thymidine kinase(HSV-tk) gene, integrated randomly into the host genome. Such exogenousdefective genes were then specifically repaired by homologousrecombination with newly introduced exogenous DNA carrying the same genebearing a different mutation. Productive gene targeting events wereidentified by selection for cells with the wild type phenotype, eitherby resistance to the drug G418 (neo^(r)) or ability to grow in HATmedium (tk⁺). See, e.g., Folger, K. R., et al. (1984), Cold SpringHarbor Symp. Quant. Biol., 49, 123-138; Lin, F. L. et al. (1984), ColdSpring Harbor Symp. Quant. Biol., 49, 139-149; Smithies, O., et al.(1984), Cold Spring Harbor Symp. Quant. Biol., 49, 161-170; Smith, A. J.H., et al. (1984), Cold Spring Harbor Symp. Quant. Biol., 49, 171-181;Thomas K. R., et al. (1986), Cell, 41, 419-428; Thomas, K. R., et al.(1986), Nature, 324, 34-38; Doetschman, T., et al. (1987), Nature, 330,576-578; and Song, Kuy-Young, et al. (1987), Proc. Natl. Acad. Sci.U.S.A., 84, 6820-6824. A similar approach has been used in plant cellswhere partially deleted neomycin resistance genes reportedly wererandomly inserted into the genome of tobacco plants. Transformation withvectors containing the deleted sequences conferred resistance toneomycin in those plant cells wherein homologous recombination occurred.Paszkowski, J., et al. (1988), EMBO J., 7, 4021-4026.

A specific requirement and significant limitation to this approach isthe necessity that the targeted gene confer a positive selectioncharacteristic in those cells wherein homologous recombination hasoccurred. In each of the above cases, a defective exogenous positiveselection marker was inserted into the genome. Such a requirementseverely limits the utility of such systems to the detection ofhomologous recombination events involving inserted selectable genes.

In a related approach, Thomas, K. R., et al. (1987), Cell, 51, 503-512,report the disruption of a selectable endogenous mouse gene byhomologous recombination. In this approach, a vector was constructedcontaining a neomycin resistance gene inserted into sequences encodingan exon of the mouse hypoxanthine phosphoribosyl transferase (Hprt)gene. This endogenous gene was selected for two reasons. First, the Hprtgene lies on the X-chromosome. Since embryonic stem cells (ES cells)derived from male embryos are hemizygous for Hprt, only a single copy ofthe Hprt gene need be inactivated by homologous recombination to producea selectable phenotype. Second, selection procedures are available forisolating Hprt⁻ mutants. Cells wherein homologous recombination eventsoccurred could thereafter be positively selected by detecting cellsresistant to neomycin (neo^(R)) and 6-thioguanine (Hprt⁻).

A major limitation in the above methods has been the requirement thatthe target sequence in the genome, either endogenous or exogenous,confer a selection characteristic to the cells in which homologousrecombination has occurred (i.e. neo^(R), tk⁺ or Hprt⁻). Further, forthose gene sequences which confer a selectable phenotype upon homologousrecombination (e.g. the Hprt gene), the formation of such a selectablephenotype requires the disruption of the endogenous gene.

The foregoing approaches to gene targeting are clearly not applicable tomany emerging technologies. See, e.g. Friedman, T. (1989), Science, 244,1275-1281 (human gene therapy); Gasser, C. S., et al., Id., 1293-1299(genetic engineering of plants); Pursel, I. G., et al., Id. 1281-1288(genetic engineering of livestock); and Timberlake, W. E., et al., Id.et al., 13-13, 1312 (genetic engineering of filamentous fungi). Suchtechniques are generally not useful to isolate transformants whereinnon-selectable endogenous genes are disrupted or modified by homologousrecombination. The above methods are also of little or no use for genetherapy because of the difficulty in selecting cells wherein the geneticdefect has been corrected by way of homologous recombination.

Recently, several laboratories have reported the expression of anexpression-defective exogenous selection marker after homologousintegration into the genome of mammalian cells. Sedivy, J. M., et al.(1989), Proc. Nat. Acad. Sci. U.S.A., 86, 227-231, report targeteddisruption of the hemizygous polyomavirus middle-T antigen with aneomycin resistance gene lacking an initiation codon. Successfultransformants were selected for resistance to G418. Jasin, M., et al.(1988), Genes and Development, 2, 1353-1363 report integration of anexpression-defective gpt gene lacking the enhancer in its SV40 earlypromotor into the SV40 early region of a gene already integrated intothe mammalian genome. Upon homologous recombination, the defective gptgene acts as a selectable marker.

Assays for detecting homologous recombination have also recently beenreported by several laboratories. Kim, H. S., et al. (1988), Nucl. Acid.S. Res., 16, 8887-8903, report the use of the polymerase chain reaction(PCR) to identify the disruption of the mouse hprt gene. A similarstrategy has been used by others to identify the disruption of the Hox1.1 gene in mouse ES cells (Zimmmer, A. P., et al. (1989), Nature, 338,150-153) and the disruption of the En-2 gene by homologous recombinationin embryonic stem cells. (Joyner, A. L., et al. (1989), Nature, 338,153-156).

It is an object herein to provide methods whereby any predeterminedregion of the genome of a cell or organism may be modified and whereinsuch modified cells can be selected and enriched.

It is a further object of the invention to provide novel vectors used inpracticing the above methods of the invention.

Still further, an object of the invention is to provide transformedcells which have been modified by the methods and vectors of theinvention to contain desired mutations in specific regions of the genomeof the cell.

Further, it is an object herein to provide non-human transgenicorganisms, which contain cells having predetermined genomicmodifications.

The references discussed above are provided solely for their disclosureprior to the filing date of the present application. Nothing herein isto be construed as an admission that the inventors are not entitled toantedate such disclosure by virtue of prior invention.

SUMMARY OF THE INVENTION

In accordance with the above objects, positive-negative selector (PNS)vectors are provided for modifying a target DNA sequence contained inthe genome of a target cell capable of homologous recombination. Thevector comprises a first DNA sequence which contains at least onesequence portion which is substantially homologous to a portion of afirst region of a target DNA sequence. The vector also includes a secondDNA sequence containing at least one sequence portion which issubstantially homologous to another portion of a second region of atarget DNA sequence. A third DNA sequence is positioned between thefirst and second DNA sequences and encodes a positive selection markerwhich when expressed is functional in the target cell in which thevector is used. A fourth DNA sequence encoding a negative selectionmarker, also functional in the target cell, is positioned 5′ to thefirst or 3′ to the second DNA sequence and is substantially incapable ofhomologous recombination with the target DNA sequence.

The above PNS vector containing two homologous portions and a positiveand a negative selection marker can be used in the methods of theinvention to modify target DNA sequences. In this method, cells arefirst transfected with the above vector. During this transformation, thePNS vector is most frequently randomly integrated into the genome of thecell. In this case, substantially all of the PNS vector containing thefirst, second, third and fourth DNA sequences is inserted into thegenome. However, some of the PNS vector is integrated into the genomevia homologous recombination. When homologous recombination occursbetween the homologous portions of the first and second DNA sequences ofthe PNS vector and the corresponding homologous portions of theendogenous target DNA of the cell, the fourth DNA sequence containingthe negative selection marker is not incorporated into the genome. Thisis because the negative selection marker lies outside of the regions ofhomology in the endogenous target DNA sequence. As a consequence, atleast two cell populations are formed. That cell population whereinrandom integration of the vector has occurred can be selected against byway of the negative selection marker contained in the fourth DNAsequence. This is because random events occur by integration at the endsof linear DNA. The other cell population wherein gene targeting hasoccurred by homologous recombination are positively selected by way ofthe positive selection marker contained in the third DNA sequence of thevector. This cell population does not contain the negative selectionmarker and thus survives the negative selection. The net effect of thispositive-negative selection method is to substantially enrich fortransformed cells containing a modified target DNA sequence.

If in the above PNS vector, the third DNA sequence containing thepositive selection marker is positioned between first and second DNAsequences corresponding to DNA sequences encoding a portion of apolypeptide (e.g. within the exon of a eucaryotic organism) or within aregulatory region necessary for gene expression, homologousrecombination allows for the selection of cells wherein the genecontaining such target DNA sequences is modified such that it is nonfunctional.

If, however, the positive selection marker contained in the third DNAsequence of the PNS vector is positioned within an untranslated regionof the genome, e.g. within an intron in a eucaryotic gene, modificationsof the surrounding target sequence (e.g. exons and/or regulatoryregions) by way of substitution, insertion and/or deletion of one ormore nucleotides may be made without eliminating the functionalcharacter of the target gene.

The invention also includes transformed cells containing at least onepredetermined modification of a target DNA sequence contained in thegenome of the cell.

In addition, the invention includes organisms such as non-humantransgenic animals and plants which contain cells having predeterminedmodifications of a target DNA sequence in the genome of the organism.

Various other aspects of the invention will be apparent from thefollowing detailed description, appended drawings and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the positive-negative selection (PNS) vector of theinvention and a target DNA sequence.

FIGS. 2a and 2 b depict the results of gene targeting (homologousrecombination) and random integration of a PNS vector into a genome.

FIG. 3 depicts a PNS vector containing a positive selection markerwithin a sequence corresponding, in part, to an intron of a target DNAsequence.

FIG. 4 is a graphic representation of the absolute frequency ofhomologous recombination versus the amount of 100% sequence homology inthe first and second DNA sequences of the PNS vectors of the invention.

FIGS. 5a, 5 b, 5 c and 5 d depict the construction of a PNS vector usedto disrupt the INT-2 gene.

FIG. 6 depicts the construction of a PNS vector used to disrupt theHOX1.4 gene.

FIGS. 7A, 7B and 7C depict the construction of a PNS vector used totransform endothelial cells to express factor VIII.

FIG. 8 depicts a PNS vector to correct a defect in the purine nucleosidephosphorylase gene.

FIG. 9 depicts a vector for promoterless PNS.

FIG. 10 depicts the construction of a PNS vector to target an induciblepromoter into the int-2 locus.

DETAILED DESCRIPTION OF THE INVENTION

The positive-negative selection (“PNS”) methods and vectors of theinvention are used to modify target DNA sequences in the genome of cellscapable of homologous recombination.

A schematic diagram of a PNS vector of the invention is shown in FIG. 1.As can be seen, the PNS vector comprises four DNA sequences. The firstand second DNA sequences each contain portions which are substantiallyhomologous to corresponding homologous portions in first and secondregions of the targeted DNA. Substantial homology is necessary betweenthese portions in the PNS vector and the target DNA to insure targetingof the PNS vector to the appropriate region of the genome.

As used herein, a “target DNA sequence” is a predetermined region withinthe genome of a cell which is targeted for modification by the PNSvectors of the invention. Target DNA sequences include structural genes(i.e., DNA sequences encoding polypeptides including in the case ofeucaryots, introns and exons), regulatory sequences such as enhancerssequences, promoters and the like and other regions within the genome ofinterest. A target DNA sequence may also be a sequence which, whentargeted by a vector has no effect on the function of the host genome.Generally, the target DNA contains at least first and second regions.See FIG. 1. Each region contains a homologous sequence portion which isused to design the PNS vector of the invention. In some instances, thetarget DNA sequence also includes a third and in some cases a third andfourth region. The third and fourth regions are substantially contiguouswith the homologous portions of the first and second region. Thehomologous portions of the target DNA are homologous to sequenceportions contained in the PNS vector. The third and in some cases thirdand fourth regions define genomic DNA sequences within the target DNAsequence which are not substantially homologous to the fourth and insome cases fourth and fifth DNA sequences of the PNS vector.

Also included in the PNS vector are third and fourth DNA sequences whichencode respectively “positive” and “negative” selection markers.Examples of preferred positive and negative selection markers are listedin Table I. The third DNA sequence encoding the positive selectionmarker is positioned between the first and second DNA sequences whilethe fourth DNA sequence encoding the negative selection marker ispositioned either 3′ to the second DNA sequences shown in FIG. 1, or 5′to the first DNA sequence (not shown in FIG. 1). The positive andnegative selection markers are chosen such that they are functional inthe cells containing the target DNA.

Positive and/or negative selection markers are “functional” intransformed cells if the phenotype expressed by the DNA sequencesencoding such selection markers is capable of conferring either apositive or negative selection characteristic for the cell expressingthat DNA sequence. Thus, “positive selection” comprises contacting cellstransfected with a PNS vector with an appropriate agent which kills orotherwise selects against cells not containing an integrated positiveselection marker. “Negative selection” on the other hand comprisescontacting cells transfected with the PNS vector with an appropriateagent which kills or otherwise selects against cells containing thenegative selection marker. Appropriate agents for use with specificpositive and negative selection markers and appropriate concentrationsare listed in Table I. Other positive selection markers include DNAsequences encoding membrane bound polypeptides. Such polypeptides arewell known to those skilled in the art and contain a secretory sequence,an extracellular domain, a transmembrane domain and an intracellulardomain. When expressed as a positive selection marker, such polypeptidesassociate with the target cell membrane. Fluorescently labelledantibodies specific for the extracellular domain may then be used in afluoresence activated cell sorter (FACS) to select for cells expressingthe membrane bound polypeptide. FACS selection may occur before or afternegative selection.

TABLE I Selectable Markers for Use in PNS-Vectors Preferred Concentra-tion of Selective selective Gene Type Agents Agent Organism Neo + G41850-1000 μg/ml Eukaryotes Neo + Kanamycin 5-500 μg/ml Plants Hyg +Hygromycin 10-1000 μg/ml Eukaryotes hisD + Histidinol 5-500 μg/mlAnimals Gpt + Xanthine, 50-500 μg/ml Animals Ble + Bleomycin 1-100 μg/mlPlants Hprt + Hypoxanthine 0.01-10 mM All HSV-tk − Acyclovir 1-100 μMAnimals Gancyclovir 0.05-200 μM Animals FIAU 0.02-100 μM Animals Hprt −6-thioguanine 0.1-100 μg/ml All Gpt − 6-thioxanthine 0.1-100 μg/mlAnimals Diphtheria − None None Animals toxin Ricin toxin − None NoneAnimals cytosine − 5-fluoro- 10-500 μg/ml All deaminase cytosine

The expression of the negative selection marker in the fourth DNAsequence is generally under control of appropriate regulatory sequenceswhich render its expression in the target cell independent of theexpression of other sequences in the PNS vector or the target DNA. Thepositive selection marker in the third DNA, however, may be constructedso that it is independently expressed (eg. when contained in an intronof the target DNA) or constructed so that homologous recombination willplace it under control of regulatory sequences in the target DNAsequence. The strategy and details of the expression of the positiveselection marker will be discussed in more detail hereinafter.

The positioning of the negative selection marker as being either “5′” or“3′” is to be understood as relating to the positioning of the negativeselection marker relative to the 5′ or 3′ end of one of the strands ofthe double-stranded PNS vector. This should be apparent from FIG. 1. Thepositioning of the various DNA sequences within the PNS vector, however,does not require that each of the four DNA sequences betranscriptionally and translationally aligned on a single strand of thePNS vector. Thus, for example, the first and second DNA sequences mayhave a 5′ to 3′ orientation consistent with the 5′ to 3′ orientation ofregions 1 and 2 in the target DNA sequence. When so aligned, the PNSvector is a “replacement PNS vector” upon homologous recombination thereplacement PNS vector replaces the genomic DNA sequence between thehomologous portions of the target DNA with the DNA sequences between thehomologous portion of the first and second DNA sequences of the PNSvector. Sequence replacement vectors are preferred in practicing theinvention. Alternatively, the homologous portions of the first andsecond DNA sequence in the PNS vector may be inverted relative to eachother such that the homologous portion of DNA sequence 1 corresponds 5′to 3′ with the homologous portion of region 1 of the target DNA sequencewhereas the homologous portion of DNA sequence 2 in the PNS vector hasan orientation which is 3′ to 5′ for the homologous portion of thesecond region of the second region of the target DNA sequence. Thisinverted orientation provides for and “insertion PNS vector”. When aninsertion PNS vector is homologously inserted into the target DNAsequence, the entire PNS vector is inserted into the target DNA sequencewithout replacing the homologous portions in the target DNA. Themodified target DNA so obtained necessarily contains the duplication ofat least those homologous portions of the target DNA which are containedin the PNS vector. Sequence replacement vectors and sequence insertionvectors utilizing a positive selection marker only are described byThomas et al. (1987), Cell, 51, 503-512.

Similarly, the third and fourth DNA sequences may be transcriptionallyinverted relative to each other and to the transcriptional orientationof the target DNA sequence. This is only the case, however, whenexpression of the positive and/or negative selection marker in the thirdand/or fourth DNA sequence respectively is independently controlled byappropriate regulatory sequences. When, for example a promoterlesspositive selection marker is used as a third DNA sequence such that itsexpression is to be placed under control of an endogenous regulatoryregion, such a vector requires that the positive selection marker bepositioned so that it is in proper alignment (5′ to 3′ and properreading frame) with the transcriptional orientation and sequence of theendogenous regulatory region.

Positive-negative selection requires that the fourth DNA sequenceencoding the negative marker be substantially incapable of homologousrecombination with the target DNA sequence. In particular, the fourthDNA sequence should be substantially non-homologous to a third region ofthe target DNA. When the fourth DNA sequence is positioned 3′ to thesecond DNA sequence, the fourth DNA sequence is non-homologous to athird region of the target DNA which is adjacent to the second region ofthe target DNA. See FIG. 1. When the fourth DNA sequence is located 5′to the first DNA sequence, it is non-homologous to a fourth region ofthe target DNA sequence adjacent to the first region of the target DNA.

In some cases, the PNS vector of the invention may be constructed with afifth DNA sequence also encoding a negative selection marker. In suchcases, the fifth DNA sequence is positioned at the opposite end of thePNS vector to that containing the fourth DNA sequence. The fourth DNAsequence is substantially non-homologous to the third region of thetarget DNA and the fifth DNA sequence is substantially non-homologous tothe fourth region of the target DNA. The negative selection markerscontained in such a PNS vector may either be the same or differentnegative selection markers. When they are different such that theyrequire the use of two different agents to select again cells containingsuch negative markers, such negative selection may be carried outsequentially or simultaneously with appropriate agents for the negativeselection marker. The positioning of two negative selection markers atthe 5′ and 3′ end of a PNS vector further enhances selection againsttarget cells which have randomly integrated the PNS vector. This isbecause random integration sometimes results in the rearrangement of thePNS vector resulting in excision of all or part of the negativeselection marker prior to random integration. When this occurs, cellsrandomly integrating the PNS vector cannot be selected against. However,the presence of a second negative selection marker on the PNS vectorsubstantially enhances the likelihood that random integration willresult in the insertion of at least one of the two negative selectionmarkers.

The substantial non-homology between the fourth DNA sequence (and insome cases fourth and fifth DNA sequences) of the PNS vector and thetarget DNA creates a discontinuity in sequence homology at or near thejuncture of the fourth DNA sequence. Thus, when the vector is integratedinto the genome by way of the homologous recombination mechanism of thecell, the negative selection marker in the fourth DNA sequence is nottransferred into the target DNA. It is the non-integration of thisnegative selection marker during homologous recombination which formsthe basis of the PNS method of the invention.

As used herein, a “modifying DNA sequence” is a DNA sequence containedin the first, second and/or third DNA sequence which encodes thesubstitution, insertion and/or deletion of one or more nucleotides inthe target DNA sequence after homologous insertion of the PNS vectorinto the targeted region of the genome. When the PNS vector containsonly the insertion of the third DNA sequence encoding the positiveselection marker, the third DNA sequence is sometimes referred to as a“first modifying DNA sequence”. When in addition to the third DNAsequence, the PNS vector also encodes the further substitution,insertion and/or deletion of one or more nucleotides, that portionencoding such further modification is sometimes referred to as a “secondmodifying DNA sequence”. The second modifying DNA sequence may comprisethe entire first and/or second DNA sequence or in some instances maycomprise less than the entire first and/or second DNA sequence. Thelatter case typically arises when, for example, a heterologous gene isincorporated into a PNS vector which is designed to place thatheterologous gene under the regulatory control of endogenous regulatorysequences. In such a case, the homologous portion of, for example, thefirst DNA sequence may comprise all or part of the targeted endogenousregulatory sequence and the modifying DNA sequence comprises thatportion of the first DNA sequence (and in some cases a part of thesecond DNA sequence as well) which encodes the heterologous DNAsequence. An appropriate homologous portion in the second DNA sequencewill be included to complete the targeting of the PNS vector. On theother hand, the entire first and/or second DNA sequence may comprise asecond modifying DNA sequence when, for example, either or both of theseDNA sequences encode for the correction of a genetic defect in thetargeted DNA sequence.

As used herein, “modified target DNA sequence” refers to a DNA sequencein the genome of a targeted cell which has been modified by a PNSvector. Modified DNA sequences contain the substitution, insertionand/or deletion of one or more nucleotides in a first transformed targetcell as compared to the cells from which such transformed target cellsare derived. In some cases, modified target DNA sequences are referredto as “first” and/or “second modified target DNA sequences”. Thesecorrespond to the DNA sequence found in the transformed target cell whena PNS vector containing a first or second modifying sequence ishomologously integrated into the target DNA sequence.

“Transformed target cells” sometimes referred to as “first transformedtarget cells” refers to those target cells wherein the PNS vector hasbeen homologously integrated into the target cell genome. A “transformedcell” on the other hand refers to a cell wherein the PNS hasnon-homologously inserted into the genome randomly. “Transformed targetcells” generally contain a positive selection marker within the modifiedtarget DNA sequence. When the object of the genomic modification is todisrupt the expression of a particular gene, the positive selectionmarker is generally contained within an exon which effectively disruptstranscription and/or translation of the targeted endogenous gene. When,however, the object of the genomic modification is to insert anexogenous gene or correct an endogenous gene defect, the modified targetDNA sequence in the first transformed target cell will in additioncontain exogenous DNA sequences or endogenous DNA sequencescorresponding to those found in the normal, i.e., nondefective,endogenous gene.

“Second transformed target cells” refers to first transformed targetcells whose genome has been subsequently modified in a predeterminedway. For example, the positive selection marker contained in the genomeof a first transformed target cell can be excised by homologousrecombination to produce a second transformed target cell. The detailsof such a predetermined genomic manipulation will be described in moredetail hereinafter.

As used herein, “heterologous DNA” refers to a DNA sequence which isdifferent from that sequence comprising the target DNA sequence.Heterologous DNA differs from target DNA by the substitution, insertionand/or deletion of one or more nucleotides. Thus, an endogenous genesequence may be incorporated into a PNS vector to target its insertioninto a different regulatory region of the genome of the same organism.The modified DNA sequence so obtained is a heterologous DNA sequence.Heterologous DNA sequences also include endogenous sequences which havebeen modified to correct or introduce gene defects or to change theamino acid sequence encoded by the endogenous gene. Further,heterologous DNA sequences include exogenous DNA sequences which are notrelated to endogenous sequences, e.g. sequences derived from a differentspecies. Such “exogenous DNA sequences” include those which encodeexogenous polypeptides or exogenous regulatory sequences. For example,exogenous DNA sequences which can be introduced into murine or bovine EScells for tissue specific expression (e.g. in mammary secretory cells)include human blood factors such as t-PA, Factor VIII, serum albumin andthe like. DNA sequences encoding positive selection markers are furtherexamples of heterologous DNA sequences.

The PNS vector is used in the PNS method to select for transformedtarget cells containing the positive selection marker and against thosetransformed cells containing the negative selection marker. Suchpositive-negative selection procedures substantially enrich for thosetransformed target cells wherein homologous recombination has occurred.As used herein, “substantial enrichment” refers to at least a two-foldenrichment of transformed target cells as compared to the ratio ofhomologous transformants versus nonhomologous transformants, preferablya 10-fold enrichment, more preferably a 1000-fold enrichment, mostpreferably a 10,000-fold enrichment, i.e., the ratio of transformedtarget cells to transformed cells. In some instances, the frequency ofhomologous recombination versus random integration is of the order of 1in 1000 and in some cases as low as 1 in 10,000 transformed cells. Thesubstantial enrichment obtained by the PNS vectors and methods of theinvention often result in cell populations wherein about 1%, and morepreferably about 20%, and most preferably about 95% of the resultantcell population contains transformed target cells wherein the PNS vectorhas been homologously integrated. Such substantially enrichedtransformed target cell populations may thereafter be used forsubsequent genetic manipulation, for cell culture experiments or for theproduction of transgenic organisms such as transgenic animals or plants.

FIGS. 2a and 2 b show the consequences of gene targeting (homologousrecombination) and random integration of a PNS vector into the genome ofa target cell. The PNS vector shown contains a neomycin resistance geneas a positive selection marker (neo^(r)) and a herpes simplex virusthymidine kinase (HSV-tk) gene as a negative selection marker. Theneo^(r) positive selection marker is positioned in an exon of gene X.This positive selection marker is constructed such that it's expressionis under the independent control of appropriate regulatory sequences.Such regulatory sequences may be endogenous to the host cell in whichcase they are preferably derived from genes actively expressed in thecell type. Alteratively, such regulatory sequences may be inducible topermit selective activation of expression of the positive selectionmarker. on each side of the neo^(r) marker are DNA sequences homologousto the regions 5′ and 3′ from the point of neo^(r) insertion in the exonsequence. These flanking homologous sequences target the X gene forhomologous recombination with the PNS vector. Consistent with the abovedescription of the PNS vector, the negative selection marker HSV-tk issituated outside one of the regions of homology. In this example it is3′ to the transcribed region of gene X. The neo^(r) gene confersresistance to the drug G418 (G418^(R)) whereas the presence of theHSV-tk gene renders cells containing this gene sensitive to gancyclovir(GANC^(s)). When the PNS vector is randomly inserted into the genome bya mechanism other than by homologous recombination (FIG. 2b), insertionis most frequently via the ends of the linear DNA and thus the phenotypefor such cells is neo⁺ HSV-tk⁺ (G418^(R), GANC^(S)). When the PNS vectoris incorporated into the genome by homologous recombination as in FIG.2a, the resultant phenotype is neo⁺, HSV-tk-(G418^(R), GANC^(R)). Thus,those cells wherein random integration of the PNS vector has occurredcan be selected against by treatment with GANC. Those remainingtransformed target cells wherein homologous recombination has beensuccessful can then be selected on the basis of neomycin resistance andGANC resistance. It, of course, should be apparent that the order ofselection for and selection against a particular genotype is notimportant and that in some instances positive and negative selection canoccur simultaneously.

As indicated, the neomycin resistance gene in FIG. 2 is incorporatedinto an exon of gene X. As so constructed, the integration of the PNSvector by way of homologous recombination effectively blocks theexpression of gene X. In multicellular organisms, however, integrationis predominantly random and occurs, for the most part, outside of theregion of the genome encoding gene X. Non-homologous recombinationtherefore will not disrupt gene X in most instances. The resultantphenotypes will therefore, in addition to the foregoing, will also be X⁻for homologous recombination and X⁺ for random integration. In manycases it is desirable to disrupt genes by positioning the positiveselection marker in an exon of a gene to be disrupted or modified. Forexample, specific proto-oncogenes can be mutated by this method toproduce transgenic animals. Such transgenic animals containingselectively inactivated proto-oncogenes are useful in dissecting thegenetic contribution of such a gene to oncogenesis and in some casesnormal development.

Another potential use for gene inactivation is disruption ofproteinaceous receptors on cell surfaces. For example, cell lines ororganisms wherein the expression of a putative viral receptor has beendisrupted using an appropriate PNS vector can be assayed with virus toconfirm that the receptor is, in fact, involved in viral infection.Further, appropriate PNS vectors may be used to produce transgenicanimal models for specific genetic defects. For example, many genedefects have been characterized by the failure of specific genes toexpress functional gene product, e.g. a and p thalassema, hemophilia,Gaucher's disease and defects affecting the production ofα-1-antitrypsin, ADA, PNP, phenylketonurea, familialhypercholesterolemia and retinoblastemia. Transgenic animals containingdisruption of one or both alleles associated with such disease states ormodification to encode the specific gene defect can be used as modelsfor therapy. For those animals which are viable at birth, experimentaltherapy can be applied. When, however, the gene defect affects survival,an appropriate generation (e.g. F0, F1) of transgenic animal may be usedto study in vivo techniques for gene therapy.

A modification of the foregoing means to disrupt gene X by way ofhomologous integration involves the use of a positive selection markerwhich is deficient in one or more regulatory sequences necessary forexpression. The PNS vector is constructed so that part but not all ofthe regulatory sequences for gene X are contained in the PNS vector 5′from the structural gene segment encoding the positive selection marker,e.g., homologous sequences encoding part of the promotor of the X gene.As a consequence of this construction, the positive selection marker isnot functional in the target cell until such time as it is homologouslyintegrated into the promotor region of gene X. When so integrated, geneX is disrupted and such cells may be selected by way of the positiveselection marker expressed under the control of the target genepromoter. The only limitation in using such an approach is therequirement that the targeted gene be actively expressed in the celltype used. Otherwise, the positive selection marker will not beexpressed to confer a positive selection characteristic on the cell.

In many instances, the disruption of an endogenous gene is undesirable,e.g., for some gene therapy applications. In such situations, thepositive selection marker comprising the third DNA sequence of the PNSvector may be positioned within an untranslated sequence, e.g. an intronof the target DNA or 5′ or 3′ untranslated regions. FIG. 3 depicts sucha PNS vector. As indicated, the first DNA sequence comprises part ofexon I and a portion of a contiguous intron in the target DNA. Thesecond DNA sequence encodes an adjacent portion of the same intron andoptionally may include all or a portion of exon II. The positiveselection marker of the third DNA sequence is positioned between thefirst and second sequences. The fourth DNA sequence encoding thenegative selection marker, of course, is positioned outside of theregion of homology. When the PNS vector is integrated into the targetDNA by way of homologous recombination the positive selection marker islocated in the intron of the targeted gene. The third DNA sequence isconstructed such that it is capable of being expressed and translatedindependently of the targeted gene. Thus, it contains an independentfunctional promotor, translation initiation sequence, translationtermination sequence, and in some cases a polyadenylation sequenceand/or one or more enhancer sequences, each functional in the cell typetransfected with the PNS vector. In this manner, cells incorporating thePNS vector by way of homologous recombination can be selected by way ofthe positive selection marker without disruption of the endogenous gene.Of course, the same regulatory sequences can be used to control theexpression of the positive selection marker when it is positioned withinan exon. Further, such regulatory sequences can be used to controlexpression of the negative selection marker. Regulatory sequences usefulin controlling the expression of positive and/or negative selectionmarkers are listed in Table IIB. Of course, other regulatory sequencesmay be used which are known to those skilled in the art. In each case,the regulatory sequences will be properly aligned and, if necessary,placed in proper reading frame with the particular DNA sequence to beexpressed. Regulatory sequence, e.g. enhancers and promoters fromdifferent sources may be combined to provide modulated gene expression.

TABLE IIA Tissue Specific Regulatory Sequences Cell/ Promoter/ TissueEnhancer Reference Adrenal PNMT Baetge, et al. (1988) PNAS 85 Erythoroidβ-globin Townes et al. (1985) EMBO J 4:1715 Lens α-crystallin Overteeket al. (1985) PNAS 82:7815 Liver α-FP Krumlauf et al. (1985) MCB 5:1639Lymphoid IGμ (γ-l Yamamura et al. (1986) promoter/ PNAS 83:2152 enhancerMammary WAP Gordon et al. (1987) Bio/Tech 5:1183 Nervous MBP Tamura etal. (1989) MCB 9:3122 Pancreas (B) Insulin Hanaban (1985) Nature 315:115Pancreas Elastase Swift et al. (1984) (exocrine) Cell 38:639 PituitaryProlactin Ingraham et al. (1988) Cell 55:579 Skeletal ckm Johnson et al.(1989) Muscle MCB 9:3393 Testes Protamine Stewart et al. (1988) MCB8:1748

TABLE IIB Regulatory Sequences for Use With Positive and/or NegativeSelection Markers Regulatory Sequence Cell Type PYF441 enhancer/HSV-tkpromoter embryo-derived (pMCI-Neo control) ASV-LTR fibroblasts SV-40early variety of mammalian cells Cytomegalo virus general mammalianβ-actin general mammalian MoMuLV haemopoetic stem cells SFFV haemopoeticstem cells Mannopine synthase general plant Octapine synthase generalplant Nopaline synthase general plant Cauliflower mosiac virus 35Sgeneral plant promoter/enhancer β-phaseolin seeds “insert-7” protoplasts

A modification of the target DNA sequence is also shown in FIG. 3. Inexon I of the target DNA sequence, the sixth codon GTG is shown whichencodes valine. In the first DNA sequence of the PNS vector, the codonGAG replaces the GTG codon in exon I. This latter codon encodesglutamine. Cells selected for homologous recombination as a consequenceencode a modified protein wherein the amino acid encoded by the sixthcodon is changed from valine to glutamine.

There are, of course, numerous other examples of modifications of targetDNA sequences in the genome of the cell which can be obtained by the PNSvectors and methods of the invention. For example, endogenous regulatorysequences controlling the expression of proto-oncogenes can be replacedwith regulatory sequences such as promoters and/or enhancers whichactively express a particular gene in a specific cell type in anorganism, i.e., tissue-specific regulatory sequences. In this manner,the expression of a proto-oncogene in a particular cell type, forexample in a transgenic animal, can be controlled to determine theeffect of oncogene expression in a cell type which does not normallyexpress the proto-oncogene. Alternatively, known viral oncogenes can beinserted into specific sites of the target genome to bring abouttissue-specific expression of the viral oncogene. Examples of preferredtissue-specific regulatory sequences are listed in Table IIA. Examplesof proto-oncogenes which may be modified by the PNS vectors and methodsto produce tissue specific expression and viral oncogenes which may beplaced under control of endogenous regulatory sequences are listed inTable IIIA and IIIB, respectively.

TABLE IIIA Proto-oncogenes involved in human tumors Gene Disease c-ablchronic myelogenous leukemia c-erbB squamous cell carcinoma glialblastoma c-myc Burkitt's lymphoma small cell carcinoma of lung carcinomaof breast L-myc small cell carcinoma of lung N-myc small cell carcinomaof lung neuroblastoma neu carcinoma of breast C-ras variety

TABLE IIIB Viral oncogenes known to cause tumors when ectopicallyexpressed in mice Ha-ras Sv40Tag HPV-E6 v-abl HPV-E7 v-fps PyTag v-mycv-src

As indicated, the positive-negative selection methods and vectors of theinvention are used to modify target DNA sequences in the genome oftarget cells capable of homologous recombination. Accordingly, theinvention may be practiced with any cell type which is capable ofhomologous recombination. Examples of such target cells include cellsderived from vertebrates including mammals such as humans, bovinespecies, ovine species, murine species, simian species, and othereucaryotic organisms such as filamentous fungi, and higher multicellularorganisms such as plants. The invention may also be practiced with lowerorganisms such as gram positive and gram negative bacteria capable ofhomologous recombination. However, such lower organisms are notpreferred because they generally do not demonstrate significantnon-homologous recombination, i.e., random integration. Accordingly,there is little or no need to select against non-homologoustransformants.

In those cases where the ultimate goal is the production of a non-humantransgenic animal, embryonic stem cells (ES cells) are preferred targetcells. Such cells have been manipulated to introduce transgenes. EScells are obtained from pre-implantation embryos cultured in vitro.Evans, M. J., et al. (1981), Nature, 292, 154-156; Bradley, M. O., etal. (1984), Nature, 309, 255-258; Gossler, et al. (1986), Proc. Natl.Acad. Sci. USA, 83, 9065-9069; and Robertson, et al. (1986), Nature,322, 445-448. PNS vectors can be efficiently introduced into the EScells by electroporation or microinjection or other transformationmethods, preferably electroporation. Such transformed ES cells canthereafter be combined with blastocysts from a non-human animal. The EScells thereafter colonize the embryo and can contribute to the germ lineof he resulting chimeric animal. For review see Jaenisch, R. (1988),Science, 240, 1468-1474. In the present invention, PNS vectors aretargeted to a specific portion of the ES cell genome and thereafter usedto generate chimeric transgenic animals by standard techniques.

When the ultimate goal is gene therapy to correct a genetic defect in anorganism such as a human being, the cell type will be determined by theetiology of the particular disease and how it is manifested. Forexample, hemopoietic stem cells are a preferred cells for correctinggenetic defects in cell types which differentiate from such stem cells,e.g. erythrocytes and leukocytes. Thus, genetic defects in globin chainsynthesis in erythrocytes such as sickle cell anemia, β-thalassemia andthe like may be corrected by using the PNS vectors and methods of theinvention with hematopoietic stem cells isolated from an affectedpatient. For example, if the target DNA in FIG. 3 is the sickle-cellβ-globin gene contained in a hematopoietic stem cell and the PNS vectorin FIG. 3 is targeted for this gene with the modification shown in thesixth codon, transformed hematopoietic stem cells can be obtainedwherein a normal β-globin will be expressed upon differentiation. Aftercorrection of the defect, the hematopoietic stem cells may be returnedto the bone marrow or systemic circulation of the patient to form asubpopulation of erythrocytes containing normal hemoglobin.Alternatively, hematopoietic stem cells may be destroyed in the patientby way of irradiation and/or chemotherapy prior to reintroduction of themodified hematopoietic stem cell thereby completely rectifying thedefect.

Other types of stem cells may be used to correct the specific genedefects associated with cells derived from such stem cells. Such otherstem cells include epithelial, liver, lung, muscle, endothelial,menchymal, neural and bone stem cells. Table IV identifies a number ofknown genetic defects which are amenable to correction by the PNSmethods and vectors of the invention.

Alternatively, certain disease states can be treated by modifying thegenome of cells in a way which does not correct a genetic defect per sebut provides for the supplementation of the gene product of a defectivegene. For example, endothelial cells are preferred as targets for humangene therapy to treat disorders affecting factors normally present inthe systemic circulation. In model studies using both dogs and pigsendothelial cells have been shown to form primary cultures, to betransformable with DNA in culture, and to be capable of expressing atransgene upon re-implantation in arterial grafts into the hostorganism. Wilson, et al. (1989), Science, 244, 1344; Nabel, et al.(1989), Science, 244, 1342. Since endothelial cells form an integralpart of the graft, such transformed cells can be used to produceproteins to be secreted into the circulatory system and thus serve astherapeutic agents in the treatment of genetic disorders affectingcirculating factors. Examples of such diseases include insulin-deficientdiabetes, α-1-antitrypsin deficiency, and hemophilia. Epithelial cellsprovide a particular advantage in the treatment of factor VIII-deficienthemophilia. These cells naturally produce von Willebrand factor and ithas been shown that production of active factor VIII is dependant uponthe autonomous synthesis of vWF (Toole, et al. (1986), Proc. Natl. Acad.Sci. USA, 83, 5939).

As indicated in Example 4, human endothelial cells from a hemophiliacpatient deficient in Factor VIII are modified by a PNS vector to producean enriched population of transformed endothelial cells wherein theexpression of DNA sequences encoding a secretory form of Factor VIII isplaced under the control of the regulatory sequences of the endogenousβ-actin gene. Such transformed cells are implanted into vascular graftsfrom the patient. After incorporation of transformed cells, it isgrafted back into the vascular system of the patient. The transformedcells secrete Factor XIII into the vascular system to supplement thedefect in the patients blood clotting system.

Other diseases of the immune and/or the circulatory system arecandidates for human gene therapy. The target tissue, bone marrow, isreadily accessible by current technology, and advances are being made inculturing stem cells in vitro. The immune deficiency diseases caused bymutations in the enzymes adenosine deaminase (ADA) and purine nucleotidephosphorylase (PNP), are of particular interest. Not only have the genesbeen cloned, but cells corrected by PNS gene therapy are likely to havea selective advantage over their mutant counterparts. Thus, ablation ofthe bone marrow in recipient patients may not be necessary.

The PNS approach is applicable to genetic disorders with the followingcharacteristics: first, the DNA sequence and preferably the clonednormal gene must be available; second, the appropriate, tissue relevant,stem cell or other appropriate cell must be available. Below is Table IVlisting some of the known genetic diseases, the name of the cloned gene,and the tissue type in which therapy may be appropriate. These and othergenetic disease amenable to the PNS methods and vectors of the inventionhave been reviewed. See Friedman (1989), Science, 244, 1275; Nichols, E.K. (1988), Human Gene Therapy (Harvard University Press); and ColdSprings Harbor Symposium on Quantitative Biology, Vol. 11 (1986), “TheBiology of Homo Sapiens” (Cold Springs Harbor Press).

TABLE IV Human Genetic Diseases in Which the Disease Locus has beenCloned Target Disease Gene Tissue α1-anti-trypsin α1-anti trypsin liverdisease Gaucher Disease glucocerebrosidase bone marrow Granulocyte ActinGranulocyte Actin bone marrow Deficiency Immunodeficiency Adenosinedeaminase bone marrow Immunodeficiency Purine nucleoside bone marrowMuscular most likely skeletal Dystrophy dystropin gene musclePhenylketonuria Phenylalanine liver hydroxylase Sickle Cell β-globinbone marrow Anemia Thalassemia globin bone marrow Hemophilia variousclotting bone marrow/ factors endothelial cells Familial hyper- lowdensity liver/endo- cholesterolemia lipoprotein endothelial receptorcells

As indicated, genetic defects may be corrected in specific cell lines bypositioning the positive selection marker (the second DNA sequence inthe PNS vector) in an untranslated region such as an intron near thesite of the genetic defect together with flanking segments to correctthe defect. In this approach, the positive selection marker is under itsown regulatory control and is capable of expressing itself withoutsubstantially interfering with the expression of the targeted gene. Inthe case of human gene therapy, it may be desirable to introduce onlythose DNA sequences which are necessary to correct the particulargenetic defect. In this regard, it is desirable, although not necessary,to remove the residual positive selection marker which remains aftercorrection of the genetic defect by homologous recombination.

The removal of a positive selection marker from a genome in whichhomologous insertion of a PNS vector has occurred can be accomplished inmany ways. For example, the PNS vector can include a second negativeselection marker contained within the second DNA sequence. This secondnegative selection marker is different from the first negative selectionmarker contained in the fourth DNA sequence. After homologousintegration, a second modified target DNA sequence is formed containingthe third DNA encoding both the positive selection marker and the secondnegative selection marker. After isolation and purification of the firsttransformed target cells by way of negative selection againsttransformed cells containing the first negative selection marker and forthose cells containing the positive selection marker, the firsttransformed target cells are subjected to a second cycle of homologousrecombination. In this second cycle, a second homologous vector is usedwhich contains all or part of the first and second DNA sequence of thePNS vector (encoding the second modification in the target DNA) but notthose sequences encoding the positive and second negative selectionmarkers. The second negative selection marker in the first transformedtarget cells is then used to select against unsuccessful transformantsand cells wherein the second homologous vector is randomly integratedinto the genome. Homologous recombination of this second homologousvector, however, with the second modified target DNA sequence results ina second transformed target cell type which does not contain either thepositive selection marker or the second negative selection marker butwhich retains the modification encoded by the first and/or second DNAsequences. Cells which have not homologously integrated the secondhomologous vector are selected against using the second negativeselection marker.

The PNS vectors and methods of the invention are also applicable to themanipulation of plant cells and ultimately the genome of the entireplant. A wide variety of transgenic plants have been reported, includingherbaceous dicots, woody dicots and monocots. For a summary, see Gasser,et al. (1989), Science, 244, 1293-1299. A number of different genetransfer techniques have been developed for producing such transgenicplants and transformed plant cells. One technique used Agrobacteriumtumefaciens as a gene transfer system. Rogers, et al. (1986), MethodsEnzymol., 118, 627-640. A closely related transformation utilizes thebacterium Agrobacterium rhizogenes. In each of these systems a Ti or Riplant transformation vector can be constructed containing border regionswhich define the DNA sequence to be inserted into the plant genome.These systems previously have been used to randomly integrate exogenousDNA to plant genomes. In the present invention, an appropriate PNSvector may be inserted into the plant transformation vector between theborder sequences defining the DNA sequences transferred into the plantcell by the Agrobacterium transformation vector.

Preferably, the PNS vector of the invention is directly transferred toplant protoplasts by way of methods analogous to that previously used tointroduce transgenes into protoplasts. See, e.g. Paszkowski, et al.(1984), EMBO J., 3, 2717-2722; Hain, et al. (1985), Mol. Gen. Genet.,199, 161-168; Shillito, et al. (1985), Bio./Technology, 3, 1099-1103;and Negrutiu, et al. (1987), Plant Mol. Bio., 8, 363-373. Alternatively,the PNS vector is contained within a liposome which may be fused to aplant protoplast (see, e.g. Deshayes, et al. (1985), EMBO J., 4,2731-2738) or is directly inserted to plant protoplast by way ofintranuclear microinjection (see, e.g. Crossway. et al. (1986), Mol. GenGenet., 202, 179-185, and Reich, et al. (1986), Bio/Technology, 4,1001-1004). Microinjection is the preferred method for transfectingprotoplasts. PNS vectors may also be microinjected into meristematicinflorenscences. De la Pena et al. (1987), Nature, 325, 274-276.Finally, tissue explants can be transfected by way of a high velocitymicroprojectile coated with the PNS vector analogous to the methods usedfor insertion of transgenes. See, e.g. Vasil (1988), Bio/Technology, 6,397; Klein, et al. (9187), Nature, 327, 70; Klein, et al. (1988), Proc.Natl. Acad. Sci. USA, 85, 8502; McCabe, et al. (1988), Bio/Technology,6, 923; and Klein, et al., Genetic Engineering, Vol 11, J. K. Setloweditor (Academic Press, N.Y., 1989). Such transformed explants can beused to regenerate for example various serial crops. Vasil (1988),Bio/Technology, 6, 397.

Once the PNS vector has been inserted into the plant cell by any of theforegoing methods, homologous recombination targets the PNS vector tothe appropriate site in the plant genome. Depending upon the methodologyused to transfect, positive-negative selection is performed on tissuecultures of the transformed protoplast or plant cell. In some instances,cells amenable to tissue culture may be excised from a transformed planteither from the F0 or a subsequent generation.

The PNS vectors and method of the invention are used to precisely modifythe plant genome in a predetermined way. Thus, for example, herbicide,insect and disease resistance may be predictably engineered into aspecific plant species to provide, for example, tissue specificresistance, e.g., insect resistance in leaf and bark. Alternatively, theexpression levels of various components within a plant may be modifiedby substituting appropriate regulatory elements to change the fatty acidand/or oil content in seed, the starch content within the plant and theelimination of components contributing to undesirable flavors in food.Alternatively, heterologous genes may be introduced into plants underthe predetermined regulatory control in the plant to produce varioushydrocarbons including waxes and hydrocarbons used in the production ofrubber.

The amino acid composition of various storage proteins in wheat andcorn, for example, which are known to be deficient in lysine andtryptophan may also be modified. PNS vectors can be readily designed toalter specific codons within such storage proteins to encode lysineand/or tryptophan thereby increasing the nutritional value of suchcrops. For example, the zein protein in corn (Pederson et al. (1982),Cell, 2, 1015) may be modified to have a higher content of lysine andtryptophan by the vectors and methods of the invention.

It is also possible to modify the levels of expression of variouspositive and negative regulatory elements controlling the expression ofparticular proteins in various cells and organisms. Thus, the expressionlevel of negative regulatory elements may be decreased by use of anappropriate promotor to enhance the expression of a particular proteinor proteins under control of such a negative regulatory element.Alternatively, the expression level of a positive regulatory protein maybe increased to enhance expression of the regulated protein or decreasedto reduce the amount of regulated protein in the cell or organism.

The basic elements of the PNS vectors of the invention have already beendescribed. The selection of each of the DNA sequences comprising the PNSvector, however, will depend upon the cell type used, the target DNAsequence to be modified and the type of modification which is desired.

Preferably, the PNS vector is a linear double stranded DNA sequence.However, circular closed PNS vectors may also be used. Linear vectorsare preferred since they enhance the frequency of homologous integrationinto the target DNA sequence. Thomas, et al. (1986), Cell, 44, 49.

In general, the PNS vector (including first, second, third and fourthDNA sequences) has a total length of between 2.5 kb (2500 base pairs)and 1000 kb. The lower size limit is set by two criteria. The first ofthese is the minimum necessary length of homology between the first andsecond sequences of the PNS vector and the target locus. This minimum isapproximately 500 bp (DNA sequence 1 plus DNA sequence 2). The secondcriterion is the need for functional genes in the third and fourth DNAsequences of the PNS vector. For practical reasons, this lower limit isapproximately 1000 bp for each sequence. This is because the smallestDNA sequences encoding known positive and negative selection markers areabout 1.0-1.5kb in length.

The upper limit to the length of the PNS vector is determined by thestate of the technology used to manipulate DNA fragments. If thesefragments are propagated as bacterial plasmids, a practical upper lengthlimit is about 25 kb; if propagated as cosmids, the limit is about 50kb, if propagated as YACs (yeast artificial chromosomes) the limitapproaches 1000 kb (Burke, et al. (1987), Science, 236, 806).

Within the first and second DNA sequences of the PNS vector are portionsof DNA sequence which are substantially homologous with sequenceportions contained within the first and second regions of the target DNAsequence. The degree of homology between the vector and target sequencesinfluences the frequency of homologous recombination between the twosequences. One hundred percent sequence homology is most preferred,however, lower sequence homology can be used to practice the invention.Thus, sequence homology as low as about 80% can be used. A practicallower limit to sequence homology can be defined functionally as thatamount of homology which if further reduced does not mediate homologousintegration of the PNS vector into the genome. Although as few as 25 bpof 100% homology are required for homologous recombination in mammaliancells (Ayares, et al. (1986), Genetics, 83, 5199-5203), longer regionsare preferred, e.g., 500 bp, more preferably, 5000 bp, and mostpreferably, 25000 bp for each homologous portion. These numbers definethe limits of the individual lengths of the first and second sequences.Preferably, the homologous portions of the PNS vector will be 100%homologous to the target DNA sequence, as increasing the amount ofnon-homology will result in a corresponding decrease in the frequency ofgene targeting. If non-homology does exist between the homologousportion of the PNS vector and the appropriate region of the target DNA,it is preferred that the non-homology not be spread throughout thehomologous portion but rather in discrete areas of the homologousportion. It is also preferred that the homologous portion of the PNSvector adjacent to the negative selection marker (fourth or fifth DNAsequence) be 100% homologous to the corresponding region in the targetDNA. This is to ensure maximum discontinuity between homologous andnon-homologous sequences in the PNS vector.

Increased frequencies of homologous recombination have been observedwhen the absolute amount of DNA sequence in the combined homologousportions of the first and second DNA sequence are increased. FIG. 4depicts the targeting frequency of the Hprt locus as a function of theextent of homology between an appropriate PNS vector and the endogenoustarget. A series of replacement (▴) and insertion () Hprt vectors wereconstructed that varied in the extent of homology to the endogenous Hprtgene. Hprt sequences in each vector were interrupted in the eighth exonwith the neomycin resistance gene. The amount of Hprt sequence 3′ to theneo gene was kept constant to the amount of Hprt sequence 5′ to the neowas varied. The absolute frequency of independent targeting events pertotal ES cells electroporated is plotted in FIG. 4 on the logarithmicscale as a function of the number of kilobases of Hprt sequencecontained within the PNS vectors. See Capecchi, M. R. (1989), Science,244, 1288-1292.

As previously indicated, the fourth DNA sequence containing the negativeselection marker should have sufficient non-homology to the target DNAsequence to prevent homologous recombination between the fourth DNAsequence and the target DNA. This is generally not a problem since it isunlikely that the negative selection marker chosen will have anysubstantial homology to the target DNA sequence. In any event, thesequence homology between the fourth DNA sequence and the target DNAsequence should be less than about 50%, most preferably less than about30%.

A preliminary assay for sufficient sequence non-homology between thefourth DNA sequence and the target DNA sequence utilizes standardhybridization techniques. For example, the particular negative selectionmarker may be appropriately labeled with a radioisotope or otherdetectable marker and used as a probe in a Southern blot analysis of thegenomic DNA of the target cell. If little or no signal is detected underintermediate stringency conditions such as 3×SSC when hybridized atabout 55° C., that negative selection marker should be functional in aPNS vector designed for homologous recombination in that cell type.However, even if a signal is detected, it is not necessarily indicativethat particular negative selection cannot be used in a PNS vectortargeted for that genome. This is because the negative selection markermay be hybridizing with a region of the genome which is not in proximitywith the target DNA sequence. Since the target DNA sequence is definedas those DNA sequences corresponding to first, second, third, and insome cases, fourth regions of the genome, Southern blots localizing theregions of the target DNA sequence may be performed. If the probecorresponding to the particular negative selection marker does nothybridize to these bands, it should be functional for PNS vectorsdirected to these regions of the genome.

Hybridization between sequences encoding the negative selection markerand the genome or target regions of a genome, however, does notnecessarily mean that such a negative selection marker will not functionin a PNS vector. The hybridization assay is designed to detect thosesequences which should function in the PNS vector because of theirfailure to hybridize to the target. Ultimately, a DNA sequence encodinga negative selection marker is functional in a PNS vector if it is notintegrated during homologous recombination regardless of whether or notit hybridizes with the target DNA.

It is also possible that high stringency hybridization can be used toascertain whether genes from one species can be targeted into relatedgenes in a different species. For example, preliminary gene therapyexperiments may require that human genomic sequences replace thecorresponding related genomic sequence in mouse cells. High stringencyhybridization conditions such as 0.1×SSC at about 68° C. can be used tocorrelate hybridization signal under such conditions with the ability ofsuch sequences to act as homologous portions in the first and second DNAsequence of the PNS vector. Such experiments can be routinely performedwith various genomic sequences having known differences in homology. Themeasure of hybridization may therefore correlate with the ability ofsuch sequences to bring about acceptable frequencies of recombination.

Table I identifies various positive and negative selection markers whichmay be used respectively in the third and fourth DNA sequences of thePNS vector together with the conditions used to select for or againstcells expressing each of the selection markers. As for animal cells suchas mouse L cells, ES cells, preferred positive selection markers includeDNA sequences encoding neomycin resistance and hygromycin resistance,most preferably neomycin resistance. For plant cells preferred positiveselection markers include neomycin resistance and bleomycin resistance,most preferably neomycin resistance.

For animal cells, preferred negative selection markers include gpt andHSV-tk, most preferably HSV-tk. For plant cells, preferred negativeselection markers include Gpt and HSV-tk. As genes responsible forbacterial and fungal pathogenesis in plants are cloned, other negativemarkers will become readily available.

As used herein, a “positive screening marker” refers to a DNA sequenceused in a phage rescue screening method to detect homologousrecombination. An example of such a positive screening marker is thesupF gene which encodes a tyrosine transfer RNA which is capable ofsuppressing amber mutations. See Smithies, et al. (1985), Nature, 317,230-234.

The following is presented by way of example and is not to be construedas a limitation on the scope of the invention.

EXAMPLE 1 Inactivation at the Int-2 Locus in Mouse ES Cells

1. PNS Vector Construction

The PNS vector, pINT-2-N/TK, is described in Mansour, et al. (1988),Nature, 336, 349. This vector was used to disrupt the proto-oncogene,INT-2, in mouse ES cells. As shown in FIG. 5c, it contains DNA sequences1 and 2 homologous to the target INT-2 genomic sequences in mouse EScells. These homologous sequences were obtained from a plasmid referredto as pAT-153 (Peters, et al. (1983), Cell, 33, 369). DNA sequence 3,the positive selection moiety of the PNS vector was the Neo gene fromthe plasmid pMCINeo described in Thomas, et al. (1987), Cell, 51, 503;DNA sequence 4, the negative selection element of the vector, was theHSV-TK gene derived from the plasmid pIC-19-R/TK which is widelyavailable in the scientific community. Plasmid pIC19R/MC1-TK (FIG. 5d)contains the HSV-TX gene engineered for expression in ES cells (Mansour,et al. (1988), Nature, 336, 348-352). The TK gene, flanked by aduplication of a mutant polyoma virus enhancer, PYF441, has beeninserted into the vector, pIC19R (Marsh, et al. (1984), Gene, 32,481-485) between the XhoI and the HindIII sites. The map of plasmidpIC19R/MC1-TK is shown in FIG. 5d. The enhancer sequence is as follows:

5′

CTCGAGCAGT GTGGTTTTCA AGAGGAAGCA AAAAGCCTCT CCACCCAGGC

CTGGAATGIT TCCACCCAAT GTCGAGCAGT GTGGTTTTGC AAGAGGAAGC

AAAAAGCCTC TCCACCCAGG CCTGGAATGT TTCCACCCAA TCTCGAG

3′

The 5′ end is an XhoI restriction enzyme site, the 3′ end is contiguouswith the HSV-TK gene. The HSV-TK sequences are from nucleotides 92-1799(McKnight (1980), Nucl. Acids. Res., 8, 5949-5964) followed at the 3′end by a HindIII linker. The plasmid pIC19R is essentially identical tothe pUC vectors, with an alternative poly-linker as shown in FIG. 5d.

Construction of the vector, pINT-2-N/TK involved five sequential stepsas depicted in FIG. 5. First, a 3,965 bp PstI fragment containing exon1b, was excised from pAT153 and inserted into the PstI site ofBluescribe° (Stratagene of LaJolla, Calif.), an Amp^(R) bacterialplasmid containing a multi-enzyme, cloning polylinker. Second, asynthetic XhoI linker of sequence

  5′              3′    \            /     GCTCGAGCGGCC     ||||||||CCGGCGAGCTCG

was inserted into the ApaI site on exon 1b. Third, the XhoI-SalINeo^(r)-fragment from pMCI Neo was inserted into the XhoI linker in exon1b. Fourth, the 3,965 bp INT-2 Pst fragment containing the Neo^(r) genewas reinserted into pAT153, to generate the plasmid pINT-2-N as shown inFIG. 5b. This plasmid also includes the third exon of the int-2 gene.Fifth, the ClaI-HindII HSV-tk fragment from pIC-19-R/TK was insertedinto Clal-HindII digested pINT2-N, creating the final product,pINT2-N/TK. This vector was linearized by digestion with ClaI prior toits introduction into ES cells.

2. Generation of ES Cells

ES cells were derived from two sources. The first source was isolationdirectly from C57B1/6 blastocysts (Evans, et al. (1981), Nature, 9,154-156) except that primary embryonic fibroblasts (Doetschman, et al.(1985), J. Embryol. Exp. Morphol., 87, 27-45) were used as feedersrather than STO cells. Briefly, 2.5 days postpregnancy mice wereovariectomized, and delayed blastocysts were recovered 4-6 days later.The blastocysts were cultured on mitomycin C-inactivated primaryembryonic fibroblasts. After blastocyst attachment and the outgrowth ofthe trophectoderm, the ICM-derived clump was picked and dispersed bytrypsin into clumps of 3-4 cells and put onto new feeders. All culturingwas carried out in DMEM plus 20% FCS and 10⁻⁴ β-mercaptoethanol. Thecultures were examined daily. After 6-7 days in culture, colonies thatstill resembled ES cells were picked, dispersed into single cells, andreplated on feeders. Those cell lines that retained the morphology andgrowth characteristic of ES cells were tested for pluripotency in vitro.These cell lines were maintained on feeders and transferred every 2-3days.

The second method was to utilize one of a number of ES cell linesisolated from other laboratories, e.g., CC1.2 described by Kuehn, et al.(1987), Nature, 326, 295. The cells were grown on mitomycinC-inactivated STO cells. Cells from both sources behaved identically ingene targeting experiments.

3. Introduction of PNS Vector pINT-2-N/TK into ES cells

The PNS vector pINT-2-N/TK was introduced into ES cells byelectroporation using the Promega Biotech X-Cell 2000. Rapidly growingcells were trypsinized, washed in DMEM, counted and resuspended inbuffer containing 20 mM HEPES (pH 7.0), 137 mM NaCl, 5 mM KCl, 0.7 mMNa₂HPO₄, 6 mM dextrose, and 0.1 MM β-mercaptoethanol. Just prior toelectroporation, the linearized recombinant vector was added.Approximately 25 μg of linearized PNS vector was mixed with 107 ES cellsin each 1 ml-cuvette.

Cells and DNA were exposed to two sequential 625 V/cm pulses at roomtemperature, allowed to remain in the buffer for 10 minutes, then platedin non-selective media onto feeder cells.

4. Selection of ES Cells Containing a Targeted Disruption of the Int-2Locus

Following two days of non-selective growth, the cells were trypsinizedand replated onto G418 (250 μg/ml) media. The positive-selection wasapplied alone for three days, at which time the cells were againtrypsinized and replated in the presence of G418 and either gancyclovir(2×10⁻⁶ M) (Syntex, Palo Alto, Calif.) or1-(2-deoxy-2-fluoro-β-D-arabino-furanosyl-5-iodouracil (F.I.A.U.)(1×10⁻⁶ M) (Bristol Myers). When the cells had grown to confluency, eachplate of cells was divided into two aliquots, one of which was frozen inliquid N₂, the other harvested for DNA analysis.

5. Formation of INT-2 Disrupted Transgenic Mice

Those transformed cells determined to be appropriately modified by thePNS vector were grown in non-selective media for 2-5 days prior toinjection into blastocysts according to the method of Bradley inTeratocarcinomas and embryonic stem cells, a practical approach, editedby E. J. Robertson, IRL Press, Oxford (1987), p. 125.

Blastocysts containing the targeted ES cells were implanted intopseudo-pregnant females and allowed to develop to term. Chimaericoffspring were identified by coat-color markers and those males showingchimaerism were selected for breeding offspring. Those offspring whichcarry the mutant allele can be identified by coat color, and thepresence of the mutant allele reaffirmed by DNA analysis by tail-blot,DNA analysis.

EXAMPLE 2 Disruption at the Hox1.4 Locus in Mouse ES Cells

Disruption of the hox1.4 locus was performed by methods similar to thosedescribed to disrupt the int-2 locus. There were two major differencesbetween these two disruption strategies. First, the PNS vector,pHOX1.4N/TK-TK2 (FIG. 6), used to disrupt the hox1.4 locus contained twonegative selection markers, i.e., a DNA sequence 5 encoding a secondnegative selection marker was included on the PNS vector at the endopposite to DNA sequence 4 encoding the first negative selection marker.DNA sequence 5 contained the tk gene isolated from HSV-type 2. Itfunctioned as a negative-selectable marker by the same method as theoriginal HSV-tk gene, but the two tk genes are 20% non-homologous. Thisnon-homology further inhibits recombination between DNA sequences 4 and5 in the vector which might have inhibited gene-targeting. The seconddifference between the int-2 and the hox1.4 disruption strategies isthat the vector pHOX1.4N/TK-TK2 contains a deletion of 1000 bp of hox1.4sequences internal to the gene, i.e., DNA sequences 1 and 2 are notcontiguous.

The HSV-tk2 sequences used in this construction were obtained frompDG504 (Swain, M. A. et al. (1983), J. Virol., 46, 1045). The structuralTK gene from pDG504 was inserted adjacent to the same promoter/enhancersequences used to express both the Neo and HSV-tk genes, to generate theplasmid pIC20H/TK2.

Construction of pHOX1.4N/TK-TK2 proceeded in five sequential steps asdepicted in FIG. 6. First a clone containing hox1.4 sequences wasisolated from a genomic A library. The A library was constructed byinserting EcoRI partially digested mouse DNA into the A-DASH®(Stratagene) cloning phage. The hox1.4 containing phage were identifiedby virtue of their homology to a synthetic oligonucleotide synthesizedfrom the published sequence of the hox1.4 locus. Tournier-Lasserve, etal. (1989), Mol. Cell Biol., 9, 2273. Second, a 9 kb SalI-SpeI fragmentcontaining the hox1.4 homeodomain was inserted into Bluescribe®. Third,a 1 kb BgIII fragment within the hox1.4 locus was replaced with theNeo^(r) gene isolated from pMCl Neo, creating the plasmid pHOX1.4N.Fourth, the XhoI-SalI fragment by HSV-tk from pIC19R/TK was insertedinto the SalI site of pHOX1.4N, generating the plasmid pHOX1.4N/TK.Fifth, the Sall-SpeI fragment from pHOX1.4N/TK was inserted into aSalI-XbaI digest of the plasmid pIC20HTK2, generating the final product,pHOX1.4N/TK/TK2. This vector was digested with SalI to form a linear PNSvector which was transfected into mouse ES cells as described inExample 1. Positive-negative selection and the method of formingtransgenic mice was also as described in Example 1. Southern blots ofsomatic cells demonstrate that the disrupted hox1.4 gene was transferredto transgenic offspring.

EXAMPLE 3 Inactivation of Other Hox Genes

The methods described in Examples 1 and 2 have also been used to disruptthe hox1.3, hox1.6, hox2.3, and int-1 loci in ES cells. The genomicsequences for each of these loci (isolated from the same -Dash librarycontaining the hox1.4 clone) were used to construct PNS vectors totarget disruption of these genes. All of these PNS vectors contain theNeo gene from pMCi-Neo as the positive selection marker and the HSV-tkand HSV-tk2 sequences as negative selection markers.

TABLE V Other Murine Developmental Genes Inactivated by PNSNeo-Insertion Locus Genomic Fragment Sequence Ref. Site hox1.3 11kbXba-HindIII Tournier-Iasserve, EcoRI-site in et al. (1989), homeo-domainMCE, 9, 2273 hox1.6 13kb partial RI Baron, et al. (1987), BglII-site inEMBO, 6, 2977 homeo-domain hox2.3 12kb BamHI Hart, et al. (1987),BglII-site in Genomics, 1, 182 homeo-domain int-1 13kb Bg1II Van Ooyenet al. XhoI-site in (1984), Cell, 39, 233 exon 2

EXAMPLE 4 Vascular Graft Supplementing Factor VIII

In this example, a functional factor VIII gene is targeted by a PNSvector to the β-actin locus in human endothelial cells. When soincorporated, the expression of factor VIII is controlled by the β-actinpromoter, a promoter known to function in nearly all somatic cells,including fibroblasts, epithelial and endothelial cells. PNS vectorconstruction is as follows: In step IA (FIG. 7A), the 13.8 kb EcoRIfragment containing the entire human β-actin gene from the λ-phage, 14TB(Leavitte, et al. (1984), Mol. Cell Bio., 4, 1961) is inserted, usingsynthetic Ecorl/XhoI adaptors, into the XhoI site of the TK vector,pIC-19-R/TK to form plasmid pBact/TK. See FIG. 7A.

In step 1 B (FIG. 7B), the 7.2 kb SalI fragment from a factor VIII cDNAclone including its native signal sequence (Kaufman, et al. (1988), JBC,263, 6352; Toole, et al. (1986), Proc. Natl. Acad. Sci. USA, 83, 5939)is inserted next to the Neo^(r) gene in a pMCI derivative plasmid. Thisplaces the neo^(r) gene (containing its own promoter/enhancer) 3′ to thepolyadenylation site of factor VIII. This plasmid is designatedpFVIII/Neo.

In step 2 (FIG. 7C), the factor VIII/Neo fragment is excised with XhoIas a single piece and inserted using synthetic XhoI/NcoI adaptors at theNcol site encompassing the met-initiation codon in pBact/TK. This codonlies in the 2nd exon of the β-actin gene, well away from the promoter,such that transcription and splicing of the mRNA is in the normalfashion. The vector so formed is designated pBact/FVIII/Neo/TK.

This vector is digested with either ClaI or HindIII which acts in thepolylinker adjacent to the TX gene. The linker vector is then introducedby electroporation into endothelial cells isolated from a hemophiliacpatient. The cells are then selected for G418 and gancyclovirresistance. Those cells shown by DNA analysis to contain the factor VIIIgene targeted to the β-actin locus or cells shown to express FVIII arethen seeded into a vascular graft which is subsequently implanted intothe patient's vascular system.

EXAMPLE 5 Replacement of a Mutant PNP Gene in Human Bone Marrow StemCells Using PNS

The genomic clone of a normal purine nucleoside phosphonylase (PNP)gene, available as a 12.4 kb, Xba-partial fragment (Williams, et al.(1984), Nucl. Acids Res, 12, 5779; Williams, et al. (1987), J. Biol.Chem. 262, 2332) is inserted at the XbaI site in the vector,pIC-19-R/TK. The neo^(r) gene from pMcI-Neo is inserted, using syntheticBamHI/XhoI linkers, into the BamHI site in intron 1 of the PNP gene. Thelinearized version of this vector (cut with ClaI) is illustrated in FIG.8.

Bone marrow stem cells from PNP patients transfected with this vectorare selected for neo^(r), gan^(r), in culture, and those cellsexhibiting replacement of the mutant gene with the vector gene aretransplanted into the patient.

EXAMPLE 6 Inactivation by Insertional Mutagenesis of the Hox 1.1 Locusin Mouse ES Cells, Using a Promoterless PNS Vector

A promoterless positive selection marker is obtained using the Neo^(R)gene, excised at its 5′ end by enzyme, EcoRI, from the plasmid,pMCI-Neo. Such a digestion removes the Neo structural gene from itscontrolling elements.

A promoterless PNS vector is used to insert the Neo gene into the Hox1/1 gene in ES cells. The Hox 1.1 gene is expressed in cultured embryocells (Colberg-Poley, et al. (1985), Nature, 314, 713) and the site ofinsertion, the second exon, lies 3′ to the promoter of the gene (Kessel,et al. (1987), PNAS, 84, 5306; Zimmer, et al. (1989), Nature, 338, 150).Expression of Neo will thus be dependent upon insertion at the Hox 1.1locus.

Vector construction is as follows:

Step 1—The neo gene, missing the transcriptional control sequences isremoved from pMCI-Neo, and inserted into the second exon of the 11 kb,FspI-KpnI fragment of Hox 1.1 (Kessel, et al. (1987), supra; Zimmer, etal. (1989), supra).

Step 2—The Hox 1.1-Neo sequences is then inserted adjacent to the HSV-tkgene is pIC19R/TK, creating the targeting vector, pHox1.1-N/TK. Thelinearized version of this vector is shown in FIG. 9 This vector iselectroporated into ES cells, which are then selected for Neo^(r),GanC^(r). The majority of cells surviving this selection are predictedto contain targeted insertions of Neo at the Hox1.1 locus.

EXAMPLE 7 Inducible Promoters

PNS vectors are used to insert novel control elements, for exampleinducible promoters, into specific genetic loci. This permits theinduction of specified proteins under the spatial and/or temporalcontrol of the investigator. In this example, the MT-1 promoter isinserted by PNS into the Int-2 gene in mouse ES cells.

The inducible promoter from the mouse metallothionein-I (MT-I) locus istargeted to the Int-2 locus. Mice generated from ES cells containingthis alteration have an Int-2 gene inducible by the presence of heavymetals. The expression of this gene in mammary cells is predicted toresult in oncogenesis and provides an opportunity to observe theinduction of the disease.

Vector construction is as follows:

Step 1—The Ecorl-BglII fragment from the MT-I gene (Palmiter, et al.(1982), Cell, 29, 701) is inserted by blunt-end ligation into the BSSHIIsite, 5′ to the Int-2 structural gene in the plasmid, pAT 153 (seediscussion of Example 1).

Step 2—The MCI-Neo gene is inserted into the AvrII site in intron 2 ofthe Int-2-MT-I construct.

Step 3—The int-2-MT-ILNeo fragment is inserted into the vector, pIC19R/TK, resulting in the construct shown in FIG. 10.

Introduction of this gene into mouse ES cells by electroporation,followed by Neo^(r), GanC^(r), selection results in cells containing theMT-I promoter inserted 5′ to the Int-2 gene. These cells are theninserted into mouse blastocysts to generate mice carrying thisparticular allele.

EXAMPLE 8 Inactivation of the ALS-II Gene in Tobacco Protoplasts by PNS

A number of herbicides function by targeting specific plant metabolicenzymes. Mutant alleles of the genes encoding these enzymes have beenidentified which confer resistance to specific herbicides. Protoplastscontaining these mutant alleles have been isolated in culture and grownto mature plants which retain the resistant phenotype (Botterman, et al.(1988), TIGS, 4, 219; Gasser, et al. (1989), Science, 244, 1293). Oneproblem with this technology is that the enzymes involved are oftenactive in multimer form, and are coded by more than one genetic locus.Thus, plants containing a normal (sensitive) allele at one locus and aresistant allele at another locus produce enzymes with mixed subunitswhich show unpredictable resistance characteristics.

In this example, the gene product of the ALS genes (acetolactatesynthase) is the target for both sulfonylurea and imidazolinoneherbicides (Lee, et al. (1987), EMBO, 7, 1241). Protoplasts resistant tothese herbicides have been isolated and shown to contain mutations inone of the two ALS loci. A 10 kb SpeI fragment of the ALS-II gene (Lee,et al. (1988), supra; Mazur, et al. (1987), Plant Phys., 85, 1110) issubcloned into the negative selection vector, pIC-19R/TK. A neo^(r)gene, engineered for expression in plant cells with regulating sequencesfrom the mannopine synthase gene for the TI plasmid is inserted into theEcoRI site in the coding region of the ALS-II. This PNS vector istransferred to the C3 tobacco cell line (Chalef, et al. (1984), Science,223, 1148),carrying a chlorsulfuronr allele in Als-I.

They are then selected for Neo^(r), GanC^(r). Those cells survivingselection are screened by DNA blots for candidates containing insertionsin the ALS-IL gene.

Having described the preferred embodiments of the present invention, itwill appear to those ordinarily skilled in the art that variousmodifications may be made to the disclosed embodiments, and that suchmodifications are intended to be within the scope of the presentinvention.

What is claimed is:
 1. A method of culturing a cell, comprisingculturing a cell having a genome comprising a modification of a targetDNA sequence, wherein the modification was introduced into the genome ofthe cell or an ancestor thereof by (a) transforming a population ofcells with a PNS vector; (b) identifying a cell having said genomecomprising the modification of the target DNA sequence by selecting forcells containing a positive selection characteristic and against cellscontaining a negative selection characteristic; (c) propagating thecell, wherein said PNS vector comprises: (1) a first homologous vectorDNA sequence capable of homologous recombination with a first region ofsaid target DNA sequence; (2) a positive selection marker DNA sequencecapable of conferring the positive selection characteristic in saidcells; (3) a second homologous vector DNA sequence capable of homologousrecombination with a second region of said target DNA sequence; and (4)a negative selection marker DNA sequence, capable of conferring thenegative selection characteristic in said cells, but substantiallyincapable of homologous recombination with said target DNA sequence;wherein the spatial order of said sequences in said PNS vector is: saidfirst homologous vector DNA sequence, said positive selection marker DNAsequence, said second homologous vector DNA sequence and said negativeselection marker DNA sequence as shown in FIG. 1; wherein the 5′-3′orientation of said first homologous vector sequence relative to saidsecond homologous vector sequence is the same as the 5′-3′ orientationof said first region relative to said second region of said targetsequence; wherein the vector is capable of modifying said target DNAsequence by homologous recombination of said first homologous vector DNAsequence with said first region of said target sequence and of saidsecond homologous vector DNA sequence with said second region of saidtarget sequence.
 2. The method of claim 1, wherein the cell is selectedfrom the group consisting of hematopoietic, epithelial, liver, lung,bone marrow, endothelial, mesenchymal, neural and muscle stem cells. 3.The method of claim 2, wherein the cell is a cell of a vertebrateanimal.
 4. The method claim 3, wherein the cell is a human cell.
 5. Themethod of claim 2, wherein the cell is a bone marrow stem cell.
 6. Themethod of claim 3, wherein the cell is a simian cell.
 7. A population ofcells comprising: (a) first cells having a genome comprising a targetsequence modified by insertion of a positive selection marker DNAsequence, wherein said target sequence is the same in each of said firstcells and said insertion is effected by homologous recombination of thetarget sequence with a PNS vector; and (b) second cells having genomesmodified by insertion of a PNS vector fragment comprising a positiveselection marker DNA sequences and a negative selection marker DNAsequence, wherein said PNS vector fragment is randomly distributed inthe genomes of said second cells; wherein said PNS vector comprises: (1)a first homologous vector DNA sequence capable of homologousrecombination with a first region of said target DNA sequence, (2) apositive selection marker DNA sequence capable of conferring a positiveselection characteristic in said cells, (3) a second homologous vectorDNA sequence capable of homologous recombination with a second region ofsaid target DNA sequence; (4) a negative selection marker DNA sequence,capable of conferring a negative selection characteristic in said cells,but substantially incapable of homologous recombination with said targetDNA sequence; wherein the spatial order of said sequences in said PNSvector is: said first homologous vector sequence, said positiveselection marker DNA sequence, said second homologous vector DNAsequence and said negative selection marker DNA sequence as shown inFIG. 1; wherein the 5′3′ orientation of said first homologous vectorsequence relative to said second homologous vector sequence is the sameas the 5′3′ orientation of said first region relative to said secondregion of said target sequence; wherein the vector is capable ofmodifying said target DNA sequence by homologous recombination of saidfirst homologous vector DNA sequence with said first region of saidtarget sequence and of said second homologous vector DNA sequence withsaid second region of said target sequence.